Protein 4.1 is a cytoskeletal protein critical for the normal shape and strength of the red cell membrane. It binds spectrin and actin to form junctional complexes that stabilize the spectrin skeleton, an attches to the cytoplasmic domains of integral membrane proteins. Erythrocyte protein 4.1 is actually a specialized member of a heterogeneous family of isoforms that arise from a single gene by highly regulated pathways of tissue specific alternative pre-mRNA splicing. Two major splicing pathways alter expression and function of protein 4.1 in erythroid progenitors; induction of the inclusion of exon 16, which encodes a critical peptide within the spectrin-actin binding domain; and suppression of the inclusion of exon 2', which removes an upstream translation start site, causing erythroid cells to produce the 80kd form, but not the 135 kd form prominent in many other tissues. Exon 16 and 17a inclusion is prominent in muscle, but not in other tissues. During the past five years, we have cloned and characterized the 350 kb genomic locus encoding protein 4.1, and initiated studies of the molecular basis for regulated splicing of exons 16,2' and 17a. We have identified critical RNA target sequences (cis elements) within exon 16, in the atypical 5' splice site, and in the first 25 bases in the upstream intron. We have also developed a cell-free splicing system that mimics the erythroid behavior of these sequences, have tentatively identified a 40 kd protein by UV crosslinking that may be a trans-acting splicing factor involved in exon 16 splicing, and have established a yeast 2 hybrid system that will facilitate identification of proteins that bind to splicing factors. We now propose to pursue studies designed to identify, isolate, molecularly clone, and functionally characterize the splicing factors responsible for the erythroid specific splicing of exon 16. We shall isolate RNA binding proteins, using the target sequences as probes, and the proteins that bind to them, using the yeast two hybrid system. The expression during, and impact on, erythropoiesis of these proteins will then be assessed. A similar sequence of studies will then be performed for exons 2' and 17a. In this manner, we hope to elucidate the regulation of protein 4.1 splicing and the physiological role of the factors that govern tissue-specific pre-mRNA splicing.